Abstract

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.

Nucleocapsid assembly function model. (A) Model for the NC assembly function, based on observations that NC-RNA binding is needed for the assembly of wild-type Gag proteins and that protein dimerization domains can replace the NC-RNA interaction for virus particle assembly purposes. As illustrated, Gag proteins, composed minimally of the capsid NTD and CTD, the SP1 spacer peptide, and NC, concentrate on RNA targets permitting the appropriate dimerization of CTDs, which in turn induces NTD oligomerization and assembly. (B) We hypothesize that replacement of NC with a readily accessible cysteine (S) residue will permit dimerization by cross-linking or oxidation, triggering the subsequent steps of the assembly process. Note that HIV-1 CA also has less accessible cysteines at CA residues 198 and 218.

Recombinant HIV proteins. The primary product of the HIV-1 gag gene is PrGag, which is N-terminally myristoylated and carries the MA, CA, NC, and p6 domains, along with spacer peptides between CA and NC (SP1) and between NC and p6 (SP2). The recombinant HIV Gag proteins are composed of the capsid domain and SP1 spacer and an N-terminal histidine tag of either 24 (his) or 37 (his2) residues, and they terminate either precisely after the SP1 spacer (his-CASP1 and his2-CASP1) or following additional glycine plus cysteine (S) residues (his-CASP1Cys and his2-CASP1Cys).

Assembly induction by cross-linking. His-CASP1Cys (A) or his-CASP1 (B) proteins at 10 μM were incubated for 60 min at 23°C (lanes 1 to 4), 30°C (lanes 5 to 8), or 37°C (lanes 9 to 12) in the absence (lanes 1, 2, 5, 6, 9, and 10) or presence (lanes 3, 4, 7, 8, 11, and 12) of 30 μM BMH. After incubations, proteins in supernatant (S) and pellet (P) fractions were collected by centrifugation, separated by SDS-PAGE, and visualized by Coomassie blue staining. Marker sizes of 119, 96, 53, 37, and 28 kDa, determined from proteins run in a separate lane, are indicated by dashes to the right of the gels. Monomer, dimer, and oligomer bands, respectively, are indicated by single, double, and triple arrows. Note that apparent monomer and dimer doublet bands in panel A appear to correspond to alternatively BMH-conjugated or cross-linked forms.

Gradient fractionation of assembly products. His-CASP1Cys (A to C) and his-CASP1 (D) proteins at 50 μM were incubated for 60 min in the presence of 250 μM diamide at 23°C (A), 30°C (B), or 37°C (C and D) and then subjected to rate centrifugation at 105,000 × g for 2 h at 4°C. Samples were collected from gradient tops (lane 1) to bottoms (lane 12), fractionated by SDS-PAGE, and visualized by immunoblotting. For panel E, E. coli 70 S ribosomes were centrifuged and detected by spectrophotometric quantitation of rRNA in gradient fractions at 260 nm. Protein monomer and dimer bands are indicated by single and double arrows, respectively. Dashes indicate the migration positions for 53- and 28-kDa marker proteins run in parallel lanes.

Morphology of his-CASP1Cys assembly products. His-CASP1Cys assembly products produced by treatment of 10 μM protein for 60 min at 23°C with 30 μM diamide followed by a 5-min incubation at 37°C were lifted onto carbon-coated EM grids, negatively stained, and imaged. Assembly products were either particles or small aggregates (bottom right of panel B and top half of panel C). Particle diameters were 136 ± 23 nm (n = 20).

Efficiency of his2-CASP1Cys assembly. His2-CASP1Cys proteins were induced to assemble by diamide treatment, fractionated into assembled pellet and unassembled supernatant fractions, separated by SDS-PAGE, and stained as described for Fig. . Band intensities from all protein species were quantitated densitometrically and used to calculate assembly efficiencies, defined as the percentage of total protein in the assembled (pellet) fraction. (A) Wild-type (WT) his2-CASP1Cys proteins were either mock treated or incubated in the presence of diamide at the indicated temperatures. (B) his2-CASP1Cys proteins either with the wild-type CA sequence or carrying NTD (Q7C) or CTD (D1696) mutations were assembled in the presence of diamide at 30°C. Assembly efficiencies were normalized to that of the WT protein.

Effects of different treatments on protein assembly. His2-CASP1Cys (A and D), his-CASP1Cys (B), and his2-CASP1 (C and E) proteins at 10 μM were incubated at 23°C for 1 h in the absence (−) or presence (+) of the following concentrations of reagents: 30 μM diamide (XL); 16.5 μg/ml of a single-stranded, 56-nt DNA oligonucleotide (DNA); 16.5 μg/ml heparin (hep); and 100 μM trypan blue (tb). After incubations, proteins in supernatant (S) and pellet (P) fractions were collected by centrifugation, subjected to SDS-PAGE on nonreducing gels, and visualized by Coomassie blue staining. Dashes on the right side of each panel indicate the migration positions of 96-, 53-, and 28-kDa marker proteins run on the same gels.

Time course of his2-CASP1Cys particle assembly. Either buffer (A) or 10 μM his2-CASP1Cys protein (B to D) samples were treated for 1 h at 23°C with 30 μM diamide prior to the addition of 1/10 volume of 10 μM fluorescein-conjugated single-stranded, 26-nt DNA oligonucleotide. As quickly as possible after DNA addition, samples were imaged by fluorescence microscopy. Note that the mock image was collected within 1.5 min of DNA addition, which appears as the quickly quenching faint haze in the upper left of panel A. Immediately after DNA addition, protein samples appeared similar to that in panel A, but within 2.5 min (B), fluorescent foci were observed, with larger fluorescent aggregates appearing on prolonged incubation (C and D).